Abstract
Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.
Highlights
Triterpene is a class of compounds in which the carbon skeleton bearing 30 carbons originated from isoprene units [1]
To select potential amino acid residues involved in the activity of friedelin synthase from M. ilicifolia (MiFRS, GenBank accession number MK526901, codon optimized for expression in Saccharomyces cerevisiae), a multiple global alignment of the primary sequences of oxidosqualene cyclase and MiFRS was performed
Increased friedelin production by the friedelin synthase mutant was observed in TwOSC1Thr502Glu
Summary
Triterpene is a class of compounds in which the carbon skeleton bearing 30 carbons originated from isoprene units [1] They play an important structural and hormonal role in plants as well as exhibiting defensive properties [2,3]. OSCs are product specific, but in plants, they compete for the same substrate to produce steroids and pentacyclic triterpenes As a result, these enzymes play a pivotal role at the border between primary and secondary metabolism [7]. The cyclization reaction of OSC requires activation by epoxide protonation, followed by Molecules 2021, 26, 6806 tacyclic triterpenes The cyclization reaction of OSC requires activa‐ tiaoncobnyceretpeodxiindterapmrolteocnualtaironat,tafoclkloowf dedoubbyleabocnodnsc,elretaediningtrtoama ochleaciur–labroaatt–tcahckairocfodnofourb-le bmonadtiso,nl,eyaideilndgintgotahechpariort–obsotearty–lcchaatironco, wnfhoircmh aistitohne,kyeiyelidnitnegrmtheediparteottospterroydluccaetisotenr,owidhailch issktehleetokne,yorintotecrhmaeird–icahteairt–ochparior dcouncfeorsmteartoioidna,lresskuellteintognin, odramtomcahreaniry–lcchaatiior–nc,hthaeirsocuonrcfeor‐ moafttiroitne,rrpeesnuelsti[n8g,9i]n(Fdiagmurme 1a)r.enyl cation, the source of triterpenes [8,9] (Figure 1)
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